Site-specific Glycoforms Research Articles

Abstract 1853: Prostate specific membrane antigen (PSMA) glycoforms and prostate cancer progression

Abstract Introduction: Prostate cancer (PCa) is the most common non-skin malignancy and the second leading cause of cancer related death in men. Prostate cancer is a heterogeneous disease with diverse clinical outcomes ranging from indolent asymptomatic cases to very aggressive and potentially lethal metastatic forms of the disease. Early detection of PCa is predominantly through determination of serum PSA levels and digital rectal examinations (DRE). Subsequently, biopsy based Gleason Grade Scores and TNM staging, tumor size or mpMRI are used to assess disease severity and progression. However, due to the low specificity of PSA concentration in diagnosis and prognosis, biopsy complications, and high mpMRI costs, there is still a critical need to identify and develop novel noninvasive biomarkers that discriminate indolent from aggressive PCa upon diagnosis. Our long-term objective is to discover biomarkers for aggressive PCa, develop and to implement them into robust clinical testing minimally invasive assays for PCa risk stratification at diagnosis. The current focus is on Prostate-Specific Membrane Antigen (PSMA), a membrane bound glycoprotein, that is significantly over-expressed in aggressive and metastatic prostate carcinomas and with poor patient prognosis. The role of its glycosylation status and composition in disease progression and its glycoforms on biomarker development and therapeutics is unknown. Aberrant glycosylation of glycoproteins occurs in many cancers, and it influences disease progression. We rationalize that differential glycosylation of PSMA correlate with progression to aggressive PCa and that PSMA glycopeptides and glycoforms can be used as diagnostics and prognosticators for aggressive disease. Methods/Results: PSMA was isolated from risk Grade Group stratified post-DRE urines, seminal plasma and prostate cancer cell lines with different metastatic phenotypes cell line lysates. Intact glycopeptides were isolated, purified and analyzed by qualitative and quantitative mass spectrometry to identify site specific glycoforms. Our data demonstrate differential expression of unique PSMA glycoforms that correlate with disease risk and severity. Conclusions: Differential glycosylation of PSMA correlates with PCa disease progression to aggressive phenotypes and may have utility in disease stratification. Ongoing studies focus on determining if these qualitative and quantitative changes have diagnostic/prognostic value and if these be integrated with other PCa biomarkers to improve their prognostic power in discriminating aggressive PCa. Acknowledgements: This work was supported by the DOD, USAMRAA, through the Prostate Cancer Research Program under Award No. W81XWH18-1-0228. Opinions, interpretations, and conclusions are those of the author and are not necessarily endorsed by the USAMRAA. Citation Format: Stephen Mackay, Ian O. Oduor, Tanya Burch, Dean A. Troyer, Oliver J. Semmes, Julius O. Nyalwidhe. Prostate specific membrane antigen (PSMA) glycoforms and prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1853.

Prostate-specific membrane antigen (PSMA) glycoforms in prostate cancer patients seminal plasma.

Prostate-specific membrane antigen (PSMA) is a USFood and Drug Administration-approved theranostic target for prostate cancer (PCa). Although PSMA is known to be glycosylated, the composition and functional roles of its N-linked glycoforms have not been fully characterized. PSMA was isolated from pooled seminal plasma from low-risk grade Groups 1 and 2 PCa patients. Intact glycopeptides were analyzed by mass spectrometry to identify site-specific glycoforms. We observed a rich distribution of PSMA glycoforms in seminal plasma from low and low-intermediate-risk PCa patients. Some interesting generalities can be drawn based on the predicted topology of PSMA on the plasma membrane. The glycoforms at ASN-459, ASN-476, and ASN-638 residues that are located at the basal domain facing the plasma membrane in cells, are predominantly high mannose glycans. ASN-76 which is located in the interdomain region adjacent to the apical domain of the protein shows a mixture of high mannose glycans and complex glycans, whereas ASN-121, ASN-195 and ASN-336 that are located and are exposed at the apical domain of the protein predominantly possess complex sialylated and fucosylated N-linked glycans. These highly accessible glycosites display the greatest diversity in isoforms across the patient samples. Our study provides novel qualitative insights into PSMA glycoforms that are present in the seminal fluid of PCa patients. The presence of a rich diversity of glycoforms in seminal plasmaprovides untapped potential for glycoprotein biomarker discovery and as a clinical sample for noninvasive diagnostics of male urological disorders and diseases including PCa. Specifically, our glycomics approach will be critical in uncovering PSMA glycoforms with utility in staging and risk stratification of PCa.

Chemical Proteomic Approach for In-Depth Glycosylation Profiling of Plasma Carcinoembryonic Antigen in Cancer Patients

Carcinoembryonic antigen (CEA) of human plasma is a biomarker of many cancer diseases, and its N-glycosylation accounts for 60% of molecular mass. It is highly desirable to characterize its glycoforms for providing additional dimension of features to increase its performance in prognosis and diagnosis of cancers. However, to systematically characterize its site-specific glycosylation is challenging because of its low abundance. Here, we developed a highly sensitive strategy for in-depth glycosylation profiling of plasma CEA through chemical proteomics combined with multienzymatic digestion. A trifunctional probe was utilized to generate covalent bond of plasma CEA and its antibody upon UV irradiation. As low as 1 ng/ml CEA in plasma could be captured and digested with trypsin and chymotrypsin for intact glycopeptide characterization. Twenty six of 28 potential N-glycosylation sites were well identified, which were the most comprehensive N-glycosylation site characterization of CEA on intact glycopeptide level as far as we known. Importantly, this strategy was applied to the glycosylation analysis of plasma CEA in cancer patients. Differential site-specific glycoforms of plasma CEA were observed in patients with colorectal cancers (CRCs) and lung cancer. The distributions of site-specific glycoforms were different as the progression of CRC, and most site-specific glycoforms were overexpressed in stage II of CRC. Overall, we established a highly sensitive chemical proteomic method to profile site-specific glycosylation of plasma CEA, which should generally applicable to other well-established cancer glycoprotein biomarkers for improving their cancer diagnosis and monitoring performance.

Glycoproteomic Analysis of Urinary Extracellular Vesicles for Biomarkers of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) accounts for the most common form of primary liver cancer cases and constitutes a major health problem worldwide. The diagnosis of HCC is still challenging due to the low sensitivity and specificity of the serum α-fetoprotein (AFP) diagnostic method. Extracellular vesicles (EVs) are heterogeneous populations of phospholipid bilayer-enclosed vesicles that can be found in many biological fluids, and have great potential as circulating biomarkers for biomarker discovery and disease diagnosis. Protein glycosylation plays crucial roles in many biological processes and aberrant glycosylation is a hallmark of cancer. Herein, we performed a comprehensive glycoproteomic profiling of urinary EVs at the intact N-glycopeptide level to screen potential biomarkers for the diagnosis of HCC. With the control of the spectrum-level false discovery rate ≤1%, 756 intact N-glycopeptides with 154 N-glycosites, 158 peptide backbones, and 107 N-glycoproteins were identified. Out of 756 intact N-glycopeptides, 344 differentially expressed intact N-glycopeptides (DEGPs) were identified, corresponding to 308 upregulated and 36 downregulated N-glycopeptides, respectively. Compared to normal control (NC), the glycoproteins LG3BP, PIGR and KNG1 are upregulated in HCC-derived EVs, while ASPP2 is downregulated. The findings demonstrated that specific site-specific glycoforms in these glycoproteins from urinary EVs could be potential and efficient non-invasive candidate biomarkers for HCC diagnosis.

Glycoproteomic Analyses of Influenza A Viruses Using timsTOF Pro MS.

N-Linked glycosylation in hemagglutinin and neuraminidase glycoproteins of influenza viruses affects antigenic and receptor binding properties, and precise analyses of site-specific glycoforms in these proteins are critical in understanding the antigenic and immunogenic properties of influenza viruses. In this study, we developed a glycoproteomic approach by using a timsTOF Pro mass spectrometer (MS) to determine the abundance and heterogeneity of site-specific glycosylation for influenza glycoproteins. Compared with a Q Exactive HF MS, the timsTOF Pro MS method without the hydrophilic interaction liquid chromatography column enrichment achieved similar glycopeptide coverage and quantities but was more effective in identifying low-abundance glycopeptides. We quantified the distributions of intact site-specific glycopeptides in hemagglutinin of A/chicken/Wuxi/0405005/2013 (H7N9) and A/mute swan/Rhode Island/A00325125/2008 (H7N3). Results showed that hemagglutinin for both viruses had complex N-glycans at N22, N38, N240, and N483 but only high-mannose glycans at N411 and, however, that the type and quantities of glycans were distinct between these viruses. Collisional cross section (CCS) provided by the ion mobility spectrometry from the timsTOF Pro MS data differentiated sialylation linkages of the glycopeptides. In summary, timsTOF Pro MS method can quantify intact site-specific glycans for influenza glycoproteins without enrichment and thus facilitate influenza vaccine development and production.

An Integrated Proteomic and Glycoproteomic Investigation Reveals Alterations in the N-Glycoproteomic Network Induced by 2-Deoxy-D-Glucose in Colorectal Cancer Cells.

As a well-known glycolysis inhibitor for anticancer treatment, 2-Deoxy-D-glucose (2DG) inhibits the growth and survival of cancer cells by interfering with the ATP produced by the metabolism of D-glucose. In addition, 2DG inhibits protein glycosylation in vivo by competing with D-mannose, leading to endoplasmic reticulum (ER) stress and unfolded protein responses in cancer cells. However, the molecular details underlying the impact of 2DG on protein glycosylation remain largely elusive. With an integrated approach to glycoproteomics and proteomics, we characterized the 2DG-induced alterations in N-glycosylation, as well as the cascading impacts on the whole proteome using the HT29 colorectal cancer cell line as a model system. More than 1700 site-specific glycoforms, represented by unique intact glycopeptides (IGPs), were identified. The treatment of 2DG had a broad effect on the N-glycoproteome, especially the high-mannose types. The glycosite occupancy of the high-mannose N-glycans decreased the most compared with the sialic acid and fucose-containing N-glycans. Many of the proteins with down-regulated high-mannose were implicated in functional networks related to response to topologically incorrect protein, integrin-mediated signaling, lysosomal transport, protein hydroxylation, vacuole, and protein N-glycosylation. The treatment of 2DG also functionally disrupted the global cellular proteome, evidenced by significant up-regulation of the proteins implicated in protein folding, endoplasmic reticulum, mitochondrial function, cellular respiration, oxidative phosphorylation, and translational termination. Taken together, these findings reveal the complex changes in protein glycosylation and expression underlying the various effects of 2DG on cancer cells, and may provide insightful clues to inform therapeutic development targeting protein glycosylation.

Integrated N-glycoproteomics Analysis of Human Saliva for Lung Cancer.

Aberrant protein N-glycosylation is a cancer hallmark, which has great potential for cancer detection. However, large-scale and in-depth analysis of N-glycosylation remains challenging because of its high heterogeneity, complexity, and low abundance. Human saliva is an attractive diagnostic body fluid, while few efforts explored its N-glycoproteome for lung cancer. Here, we utilized a zwitterionic-hydrophilic interaction chromatography-based strategy to specifically enrich salivary glycopeptides. Through quantitative proteomics analysis, 1492 and 1234 intact N-glycopeptides were confidently identified from pooled saliva samples of 10 subjects in the nonsmall-cell lung cancer group and 10 subjects in the normal control group. Accordingly, 575 and 404 N-glycosites were revealed for the lung cancer group and normal control group. In particular, 154 N-glycosites and 259 site-specific glycoforms were significantly dysregulated in the lung cancer group. Several N-glycosites located at the same glycoprotein and glycans attached to the same N-glycosites were observed with differential expressions, including haptoglobin, Mucin-5B, lactotransferrin, and α-1-acid glycoprotein 1. These N-glycoproteins were mainly related to inflammatory responses, infectious diseases, and cancers. Our study achieved comprehensive characterization of salivary N-glycoproteome, and dysregulated site-specific glycoforms hold promise for noninvasive detection of lung cancer.

Glycoforms of human prostate-specific membrane antigen (PSMA) in human cells and prostate tissue.

N-glycosylation is a ubiquitous and variable posttranslational modification that regulates physiological functions of secretory and membrane-associated proteins and the dysregulation of glycosylation pathways is often associated with cancer growth and metastasis. Prostate-specific membrane antigen (PSMA) is an established biomarker for prostate cancer imaging and therapy. Mass spectrometry was used to analyze the distribution of the site-specific glycoforms of PSMA in insect, human embryonic kidney, and prostate cancer cells, and in prostate tissue upon immunoaffinity enrichment. While recombinant PSMA expressed in insect cells was decorated mainly by paucimannose and high mannose glycans, complex, hybrid, and high mannose glycans were detected in samples from human cells and tissue. We noted an interesting spatial distribution of the glycoforms on the PSMA surface-high mannose glycans were the dominant glycoforms at the N459, N476, and N638 sequons facing the plasma membrane, while the N121, N195, and N336 sites, located at the exposed apical PSMA domain, carried primarily complex glycans. The presence of high mannose glycoforms at the former sequons likely results from the limited access of enzymes of the glycosynthetic pathway required for the synthesis of the complex structures. In line with the limited accessibility of membrane-proximal sites, no glycosylation was observed at the N51 site positioned closest to the membrane. Our study presents initial descriptive analysis of the glycoforms of PSMA observed in cell lines and in prostate tissue. It will hopefully stimulate further research into PSMA glycoforms in the context of tumor staging, noninvasive detection of prostate tumors, and the impact of glycoforms on physicochemical and enzymatic characteristics of PSMA in a tissue-specific manner.

Mirror-Cutting-Based Digestion Strategy Enables the In-Depth and Accuracy Characterization of N-Linked Protein Glycosylation.

N-linked glycosylation plays important roles in multiple physiological and pathological processes, while the analysis coverage is still limited due to the insufficient digestion of glycoproteins, as well as incomplete ion fragments for intact glycopeptide determination. Herein, a mirror-cutting-based digestion strategy was proposed by combining two orthogonal proteases of LysargiNase and trypsin to characterize the macro- and micro-heterogeneity of protein glycosylation. Using the above two proteases, the b- or y-ion series of peptide sequences were, respectively, enhanced in MS/MS, generating the complementary spectra for peptide sequence identification. More than 27% (489/1778) of the site-specific glycoforms identified by LysargiNase digestion were not covered by trypsin digestion, suggesting the elevated coverage of protein sequences and site-specific glycoforms by the mirror-cutting method. Totally, 10,935 site-specific glycoforms were identified from mouse brain tissues in the 18 h MS analysis, which significantly enhanced the coverage of protein glycosylation. Intriguingly, 27 mannose-6-phosphate (M6P) glycoforms were determined with core fucosylation, and 23 of them were found with the "Y-HexNAc-Fuc" ions after manual checking. This is hitherto the first report of M6P and fucosylation co-modifications of glycopeptides, in which the mechanism and function still needs further exploration. The mirror-cutting digestion strategy also has great application potential in the exploration of missing glycoproteins from other complex samples to provide rich resources for glycobiology research.

Automated Intact Glycopeptide Enrichment Method Facilitating Highly Reproducible Analysis of Serum Site-Specific N-Glycoproteome.

Bottom-up proteomics has been increasingly applied in clinical research to study the disease pathophysiology and to discover disease biomarkers. However, glycoproteomic analysis always requires tedious experimental steps for intact glycopeptide enrichment, which has been the technique bottleneck for large-scale analysis of clinical samples. Herein, we developed an automated glycopeptide enrichment method for the analysis of serum site-specific N-glycoproteome. This automated method allowed for processing one sample within 20 min. It showed higher enrichment specificity, more intact glycopeptide identifications, and better quantitative reproducibility than the traditional manual method using microtip enrichment devices. We further applied this method to investigate the serum site-specific N-glycosylation changes between four patients with pancreatic cancer and seven healthy controls. The principal component analysis of intact N-glycopeptides showed good clustering across cancer and normal groups. Furthermore, we found that the site-specific glycoforms, monofucosylated and nonsialylated oligosaccharides, on IgG1 site 180 expressed a significant decrease in pancreatic cancer patients compared to healthy controls. Together, the automated method is a powerful tool for site-specific N-glycoproteomic analysis of complex biological samples, and it has great potential for clinical utilities.

Analytical comparability assessment on glycosylation of ziv-aflibercept and the biosimilar candidate

Ziv-aflibercept (aflibercept) is a recombinant fusion protein which combines the portions of human vascular endothelial growth factor receptors extracellular domains fused to the Fc portion of human IgG1. It is a highly sialylated glycoprotein with 5 N-glycosylation sites. In this study, a comprehensive strategy for comparability study of the complex glycosylation was developed between aflibercept and the biosimilar candidate including the investigations on N-glycosylation sites, site occupancy, site-specific glycoforms, released glycans and sialic acids. The results indicated that same N-glycosylation sites were identified, site occupancy were 100% except N68 site, site-specific glycoforms and released glycans showed similar glycan species, contents of NANA were at a same level for two products. Minor differences were found between two products. The biosimilar candidate presented lower level of aglycosylation, lower level of glycans containing one terminal sialic acid, higher level of glycans containing two terminal sialic acids, higher level of G0F and Man5, lower level of G1F and G2F compared with aflibercept. However, further studies exhibited no differences were observed in the cell-based biological potency and Fc effector function. Moreover, the biosimilar candidate showed a similar pharmacokinetics curve and bioequivalence compared with aflibercept.

Quantitative Analysis of α-1-Antitrypsin Glycosylation Isoforms in HCC Patients Using LC-HCD-PRM-MS.

The change in glycosylation of serum proteins is often associated with the development of various diseases and thus can be used for diagnosis. In this study, a liquid chromatography-tandem mass spectrometry-based method is used for accurate structural analysis and quantification of site-specific glycoforms of serum α-1-antitrypsin (A1AT) in early-stage HCC and cirrhosis patients. Serum protein A1AT was purified from patient sera by immunoprecipitation with anti-A1AT antibody conjugated agarose beads, and the isolated A1AT protein was digested and analyzed by LC-MS/MS. Two tandem mass spectrometry strategies are integrated in this study: a nontargeted stepped HCD strategy for structural analysis of A1AT glycopeptides and a targeted parallel reaction monitoring (PRM) strategy for quantification of site-specific glycoforms of A1AT in HCC and cirrhosis patient sera. Accordingly, pGlyco2.0 software was used for glycopeptide identification, and Skyline software was used for glycoform quantification using the Y1 ion (peptide+GlcNAc) in MS/MS spectra. Ten site-specific glycopeptides of A1AT were identified with stepped HCD-MS/MS in patient samples, 7 of which were further quantified using HCD-PRM-MS among patient samples. We found that our strategy was able to distinguish isomers of glycopeptides where several isomers showed distinctly different patterns between cirrhosis and HCC patients. We also found that the ratio of different charge states (2+/3+) of one glycopeptide of A1AT can significantly discriminate early-stage HCC from cirrhosis with the area under the receiver operating characteristic curve AUC of 0.9. Further analysis showed that the difference may be related to the sialic acid/galactose linkage of the glycan motif.

Highly Efficient Analysis of Glycoprotein Sialylation in Human Serum by Simultaneous Quantification of Glycosites and Site-Specific Glycoforms.

Aberrant sialylation of glycoproteins is closely related to many malignant diseases, and analysis of sialylation has great potential to reveal the status of these diseases. However, in-depth analysis of sialylation is still challenging because of the high microheterogeneity of protein glycosylation, as well as the low abundance of sialylated glycopeptides (SGPs). Herein, an integrated strategy was fabricated for the detailed characterization of glycoprotein sialylation on the levels of glycosites and site-specific glycoforms by employing the SGP enrichment method. This strategy enabled the identification of up to 380 glycosites, as well as 414 intact glycopeptides corresponding to 383 site-specific glycoforms from only initial 6 μL serum samples, indicating the high sensitivity of the method for the detailed analysis of glycoprotein sialylation. This strategy was further employed to the differential analysis of glycoprotein sialylation between hepatocellular carcinoma patients and control samples, leading to the quantification of 344 glycosites and 405 site-specific glycoforms, simultaneously. Among these, 43 glycosites and 55 site-specific glycoforms were found to have significant change on the glycosite and site-specific glycoform levels, respectively. Interestingly, several glycoforms attached onto the same glycosite were found with different change tendencies. This strategy was demonstrated to be a powerful tool to reveal subtle differences of the macro- and microheterogeneity of glycoprotein sialylation.

Optimized Fragmentation for Quantitative Analysis of Fucosylated N-Glycoproteins by LC-MS-MRM.

Quantitative analysis of site specific glycoforms of proteins is technically challenging but highly desirable; resolution of the fucosylated glycoforms is of particular interest due to their biological importance. In this study, we developed a sensitive and specific LC-MS-MRM quantification method that distinguishes the outer arm and core fucosylated configurations of the N-glycopeptides. We take advantage of limited fragmentation of the glycopeptides at low collision energy CID to produce linkage-specific Y-ions. We select these informative ions as MRM transitions for the quantification of the outer arm and total fucosylation of 12 fucosylated glycoforms of 9 glycopeptides in 7 plasma proteins. Our workflow showed improved sensitivity and specificity of quantification of the glycopeptides compared to oxonium ion transitions which allowed us to quantify the glycoforms directly in plasma or serum without fractionation of the samples or glycopeptide enrichment. A pilot study of fucosylation in liver cirrhosis of the HCV and NASH etiologies confirms the quantitative capabilities of the method and shows that liver cirrhosis is consistently associated with increased outer arm fucosylation of majority of the analyzed proteins. The results show that the outer arm fucosylation of the A2G2F1 glycoform of the VDKDLQSLEDILHQVENK peptide of fibrinogen increases greater than 10-fold in the HCV and NASH patients compared to healthy controls.

Analysis of serum Haptoglobin using glycoproteomics and lectin immunoassay in liver diseases in Hepatitis B virus infection

BackgroundHepatocellular carcinoma (HCC) due to Hepatitis B viral (HBV) infection is a major cause in Asia-Pacific countries. Its early detection is of paramount importance using a marker having both sensitivity and specificity. The present study promises diagnostic and prognostic markers by the identification of site-specific glycoforms on Haptoglobin (Hp) using LC-MS/MS and lectin ELISA in liver diseased conditions in HBV infection. MethodsThree groups of patients: chronic, liver cirrhosis and HCC with HBV infection along with controls were enrolled. Hp was purified using affinity column chromatography and, peptide sequence, N-glycosylation site, glycan composition and glycoforms were identified using mass spectrometry. Quantitative lectin ELISA was used to measure levels of fucosylation on Hp in liver diseases due to HBV. ResultsHp levels were significantly lower in HCC when compared with Non-HCC cases (p < .05). Fucosylated glycoforms were significantly increased at site Asn184, Asn207 and Asn211 in liver diseased stages versus controls. A significant association was observed between the Fuc-Hp/Hp Elisa index and, advanced liver disease stages and controls using lectin Elisa (p < .001). ConclusionQuantitation of fucosylation levels on Hp protein using Lectin ELISA may be useful glycobiomarker either alone or in combination (AFP + DCP + FucHp; AUC = 0.94) in HBV HCC diagnosis in clinical practice.

Proteomics analysis of site-specific glycoforms by a virtual multistage mass spectrometry method

Determination of site-specific glycoforms is the key to reveal the micro-heterogeneity of protein glycosylation at proteome level. Herein, we presented an integrated virtual multistage MS strategy to identify intact glycopeptides, which allowed the determination of site-specific glycoforms. In this strategy, the enzymatically de-glycosylated peptides and intact glycopeptides were mixed and analyzed in the same LC-MS/MS run. The acquired MS2 spectra of intact glycopeptides allowed determination of the glycans, and the MS2 spectra of the de-glycosylated peptides enabled the identification of peptide backbone sequences. Compared with the conventional multistage strategy, the peptide backbones could be directly identified by the MS2 of the de-glycopeptides with higher sensitivity. This strategy was first validated by analyzing the glycosites and site-specific glycoforms of mouse liver tissues. Then, it was applied to differential analysis of the glycoproteomes of hepatocellular carcinoma (HCC) and adjacent liver tissues. Compared with the identification scheme using only MS2 spectra of intact glycopeptides or glycosites, this approach enabled quantitative analysis on two levels, i.e. glycosites and site-specific glycoforms, simultaneously. Thus, it could be a powerful tool to characterize the subtle differences in the macro- and micro-heterogeneity of protein glycosylation for different samples.

An Insight into Glyco-Microheterogeneity of Plasma von Willebrand Factor by Mass Spectrometry.

Human plasma von Willebrand Factor (VWF) plays essential roles in primary hemostasis in cooperation with other coagulations factors. There is ample indication that glycosylation affects many biological phases during the protein life cycle. However, comprehensive characterization of all probable N-glycosites simultaneous with O-glycosites is still not fully revealed. Thus, the intention of this exploration was to estimate the occupancy of all canonical N-glycosites besides simultaneous characterization of N- and O-glycoforms. An RP-LC-MS/MS system functionalized with CID and HCD tandem mass was utilized to analyze VWF. N-Glycosite occupancy varied along the protein backbone chain. Out of 257 HCD spectra, 181 characterized glycoforms were specified as either N- or O-glycosites. Sequential cleavage of glycosidic bonds along with Human Database mass matching have confirmed the glycoform structures. A total of 173 glycoforms represented most commonly biantennary and infrequently tri- and tetra-antennary N-glycans beside high mannose, hybrid, ABH antigen-terminated, and sulfated N-glycans. Many glycoforms were common across all N-sites. Noteworthy, previously unreported N-glycosites within domain D'(TIL'-E') showed glycosylation. Moreover, sialylated core 1 and core 2 O-glycans were detected on 2298T. Given subtle characterization of site-specific glycoforms, we can attain a profound understanding of the biological roles of VWF as well as facilitate the production of VWF-based therapeutics.

Adapting Data-Independent Acquisition for Mass Spectrometry-Based Protein Site-Specific N-Glycosylation Analysis.

A hallmark of protein N-glycosylation is extensive heterogeneity associated with each glycosylation site. In human cells, the constituent glycoforms differ mostly in numerous ways of extensions from an invariable trimannosyl core and terminal modifications. The efficient identification of these glycoforms at the glycopeptide level by mass spectrometry (MS) requires a precursor sampling technique that is not dictated by signal intensity or by preset targets during MS2 data acquisition. We show here that the recently developed data-independent acquisition (DIA) approach is best suited to this demanding task. It allows postacquisition extraction of glycopeptide-specific fragment-ion chromatograms to be aligned with that of precursor MS1 ion by nanoLC elution time. For any target glycoprotein, judicious selection of the most favorable MS1/MS2 transitions can first be determined from prior analysis of a purified surrogate standard that carries similar site-specific glycosylation but may differ in its exact range of glycoforms. Since the MS2 transitions to be used for extracting DIA data is common to that glycosylation site and not dictated by a specific MS1 value, our workflow applies equally well to the identification of both targeted and unexpected glycoforms. Using a case example, we show that, in targeted mode, it identified more site-specific glycoforms than the more commonly used data-dependent acquisition method when the amount of the target glycoprotein was limited in a sample of high complexity. In discovery mode, it allows detection, with supporting MS2 evidence, of under-sampled glycoforms and of those that failed to be identified by searching against a predefined glycan library owing to unanticipated modifications.

Sequential fragment ion filtering and endoglycosidase-assisted identification of intact glycopeptides.

Detailed characterization of glycoprotein structures requires determining both the sites of glycosylation as well as the glycan structures associated with each site. In this work, we developed an analytical strategy for characterization of intact N-glycopeptides in complex proteome samples. In the first step, tryptic glycopeptides were enriched using ZIC-HILIC. Secondly, a portion of the glycopeptides was treated with endoglycosidase H (Endo H) to remove high-mannose (Man) and hybrid N-linked glycans. Thirdly, a fraction of the Endo H-treated glycopeptides was further subjected to PNGase F treatment in 18O water to remove the remaining complex glycans. The intact glycopeptides and deglycosylated peptides were analyzed by nano-RPLC-MS/MS, and the glycan structures and the peptide sequences were identified by using the Byonic or pFind tools. Sequential digestion by endoglycosidase provided candidate glycosites information and indication of the glycoforms on each glycopeptide, thus helping to confine the database search space and improve the confidence regarding intact glycopeptide identification. We demonstrated the effectiveness of this approach using RNase B and IgG and applied this sequential digestion strategy for the identification of glycopeptides from the HepG2 cell line. We identified 4514 intact glycopeptides coming from 947 glycosites and 1011 unique peptide sequences from HepG2 cells. The intensity of different glycoforms at a specific glycosite was obtained to reach the occupancy ratios of site-specific glycoforms. These results indicate that our method can be used for characterizing site-specific protein glycosylation in complex samples. Graphical abstract Through integrating the information of intact glycopeptide, fragment ions filters and endoglycosidase digestion, the reliability of the identification could be significantly improved. We quantified the site-specific glycoforms occupancy ratios through the MS response signaling of each glycopeptide at the same time.

Site-specific analysis of changes in the glycosylation of proteins in liver cirrhosis using data-independent workflow with soft fragmentation.

Cirrhosis of the liver is associated with increased fucosylation of proteins in the plasma. We describe a data-independent (DIA) strategy for comparative analysis of the site-specific glycoforms of plasma glycoproteins. A library of 161 glycoforms of 25 N-glycopeptides was established by data-dependent LC-MS/MS analysis of a tryptic digest of 14 human protein groups retained on a multiple affinity removal column. The collision-induced dissociation conditions were adjusted to maximize the yield of selective Y-ions which were quantified by a data-independent mass spectrometry workflow using a 10-Da acquisition window. Using this workflow, we quantified 125 glycoforms of 25 glycopeptides, covering 10 of the 14 proteins, without any further glycopeptide enrichment. Comparison of the proteins in the plasma of healthy controls and cirrhotic patients shows an average 1.5-fold increase in the fucosylation of bi-antennary glycoforms and 3-fold increase in the fucosylation of tri- and tetra- antennary glycoforms. These results show that the adjusted glycopeptide DIA workflow using soft collision-induced fragmentation of glycopeptides is suitable for site-specific analysis of protein glycosylation in complex mixtures of analytes without glycopeptide enrichment.